Rna preparations comprising purified modified rna for reprogramming cells

Abstract

The present invention provides compositions and methods for reprogramming somatic cells using purified RNA preparations comprising single-strand mRNA encoding an iPS cell induction factor. The purified RNA preparations are preferably substantially free of RNA contaminant molecules that: i) would activate an immune response in the somatic cells, ii) would decrease expression of the single-stranded mRNA in the somatic cells, and/or iii) active RNA sensors in the somatic cells. In certain embodiments, the purified RNA preparations are substantially free of partial mRNAs, double-stranded RNAs, un-capped RNA molecules, and/or single-stranded run-on mRNAs.

We claim: 
     
         1 . A method for reprogramming a human or animal cell that exhibits a first differentiated state or phenotype to a cell that exhibits a second differentiated state or phenotype comprising: introducing into the cell that exhibits a first differentiated state a purified RNA preparation comprising modified mRNA molecules that encode at least one reprogramming factor and culturing the cell under conditions wherein the cell exhibits a second differentiated state. 
     
     
         2 . The method of  claim 1 , wherein the modified mRNA molecules contain at least one modified nucleoside selected from the group consisting of: pseudouridine (Ψ), 5-methylcytosine (m 5 C), 5-methyluridine (m 5 U), 2′-O-methyluridine (Um or m 2′-O U), 2-thiouridine (s 2 U), and N 6 -methyladenosine (m 6 A)) in place of at least a portion of the corresponding unmodified canonical nucleoside. 
     
     
         3 . The method of  claim 2 , wherein the at least one modified nucleoside is present in place of substantially all of the corresponding unmodified canonical nucleoside. 
     
     
         4 . The method of  claim 1 , wherein said purified RNA preparation:
 i) comprises at least one single-stranded mRNA that encodes an iPS cell induction factor, wherein substantially all of said first single-stranded mRNAs comprise at least one pseudouridine residue and/or at least one 5-methylcytidine residue, and   ii) is substantially free of RNA contaminant molecules which are able to activate RNA sensors in said somatic cell.   
     
     
         5 . The method of  claim 4 , wherein said RNA contaminant molecules are selected from the group consisting of: partial mRNAs encoding only a portion of said reprogramming factor, RNA molecules that are smaller than the full-length mRNA, RNA molecules that are larger than the full-length mRNA, double-stranded mRNA molecules, and un-capped mRNA molecules. 
     
     
         6 . The method of  claim 1 , wherein said cell that exhibits a second differentiated state is an iPS cell or dedifferentiated cell. 
     
     
         7 . The method of  claim 1 , wherein said cell that exhibits a second differentiated state is a transdifferentiated cell. 
     
     
         8 . The method of  claim 1 , wherein said reprogrammed cell that exhibits a second differentiated state is a redifferentiated cell. 
     
     
         9 . The method of  claim 1 , wherein said modified mRNA molecules that encode at least one reprogramming factor is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. 
     
     
         10 . The method of  claim 1 , wherein the method further comprises contacting said cell that exhibits a first differentiated state or phenotype with at least one growth factor and/or cytokine. 
     
     
         11 . The method of  claim 1 , wherein all of the uridine nucleosides in said modified mRNA are replaced by pseudouridine nucleosides. 
     
     
         12 . The method of any of  claim 1 , wherein all of the cytidine nucleosides in said modified mRNA are replaced by 5-methylcytidine nucleosides. 
     
     
         13 . The method of  claim 1 , wherein said purified RNA preparation is free of an amount of RNA contaminant molecules that would activate an immune response in said cell that exhibits a first differentiated state to prevent said cell that exhibits a second differentiated state from surviving at least 10 days in culture. 
     
     
         14 . The method of  claim 1 , wherein said cell that exhibits a second differentiated state is able to form a cell line. 
     
     
         15 . The method of  claim 14 , wherein said dedifferentiated cell expresses NANOG and TRA-1-60. 
     
     
         16 . A composition comprising a purified RNA preparation, wherein said purified RNA preparation:
 i) comprise single-stranded mRNA molecules that encode at least one reprogramming factor, wherein said single-stranded mRNA molecules comprise at least one nucleoside selected from the group consisting of pseudouridine (Ψ), 5-methylcytosine (m 5 C), 5-methyluridine (m 5 U), 2′-O-methyluridine (Um or m 2′-O U), 2-thiouridine (s 2 U), and N 6 -methyladenosine (m 6 A), and   ii) is substantially free of RNA contaminant molecules which are able to activate RNA sensors in a human or animal cell.   
     
     
         17 . The composition of  claim 16 , wherein said purified RNA preparation comprises single-stranded mRNA molecules that encode at least one reprogramming factor selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. 
     
     
         18 . A method for inducing a cell to produce the MYC protein comprising: contacting a cell with an in vitro-synthesized mRNA encoding the MYC gene, wherein the in vitro-synthesized mRNA comprises at least one pseudouridine residue and/or at least one 5-methylcytidine residue, thereby inducing the mammalian cell to produce the MYC protein.